*Disease Information: Leishmania major is a protozoan parasite, which is responsible for Leishmaniasis, a cutaneous and visceral intracellular protozoal infection. Cutaneous infections include skin rashes and sores, while visceral infections include splenomegaly and gastromegaly along with anemia. An estimated 1.3 million new cases and 20,000 to 30,000 deaths occur annually. The pathogens exist in two parasitic stages. The promastigote stage is characterized by the infection of the host by the bite of a sand-fly. Through the flagella, glycoproteins, and receptors on its membrane, the pathogen in the promastigote stage allows for the attraction of macrophages. The amastigote stage appears after the promastigote is incorporated into a macrophage. During the amastigote stage, the pathogen is present through an organism that multiplies through binary fission and spreads.
*Essentiality of this protein: Substantial research has proven 6-phospho-gluconate dehydrogenase to play a profound role in the pentose phosphate pathway, glutathione metabolism, carbon metabolism, and the biosynthesis of secondary metabolites. In vivo experiments have also concluded the inhibition of 6-phosphogluconate dehydrogenase to significantly reduce fitness in bloodstream forms in other organisms. *Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Dimer
*Complex of proteins?: Dimer
*Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8 (TDR targets link)
Structure (PDB or Homology model) -- PDB for Homology model: 1PGJ -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB: ---- Query Coverage: 96% coverage ---- Max % Identities: 73% identity ---- % Positives: 83% positive identity ---- Chain used for homology: A
Homology Model IMC:
Enter the Template PDB ID: 1PGJ
Identities 73% and Positives 57.9%
Enter the Molprobity scores for both the Template and the Model:
Current Inhibitors: Acetyl-CoA, High concentrations of palmitoyl-CoA, ATP CoA , 1-Fluoro-2,4-dinitrobenzene (irreversible), Pyridoxal 5’-phosphate with respect to ZF (competitively) and NAD or NADP (noncompetitive), L. mesenteroides G6PD, unlike mammalian G6PDs, is unaffected by steroids.
Primer design results for 'tail' primers (this is just 2 sequences): Forward:
5' TACTTCCAATCCATGTCTAACGACCTGGGTATCATTGGC 3' Reverse:
5' TATCCACCTTTACTGTTATTGCAGAGCCGGCCAT 3'
*NCBI Gene # or RefSeq#: 12980488
*Protein ID (NP or XP #) or Wolbachia#: XP_003722728.1
*Organism (including strain): Leishmania major
*Etiologic Risk Group: Risk Group 2
*Disease Information:
Leishmania major is a protozoan parasite, which is responsible for Leishmaniasis, a cutaneous and visceral intracellular protozoal infection. Cutaneous infections include skin rashes and sores, while visceral infections include splenomegaly and gastromegaly along with anemia. An estimated 1.3 million new cases and 20,000 to 30,000 deaths occur annually. The pathogens exist in two parasitic stages. The promastigote stage is characterized by the infection of the host by the bite of a sand-fly. Through the flagella, glycoproteins, and receptors on its membrane, the pathogen in the promastigote stage allows for the attraction of macrophages. The amastigote stage appears after the promastigote is incorporated into a macrophage. During the amastigote stage, the pathogen is present through an organism that multiplies through binary fission and spreads.
*Link to TDR Targets page
http://tdrtargets.org/targets/view?gene_id=23670
*Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/gene/12980488
http://www.uniprot.org/uniprot/Q9NGR0
*Essentiality of this protein:
Substantial research has proven 6-phospho-gluconate dehydrogenase to play a profound role in the pentose phosphate pathway, glutathione metabolism, carbon metabolism, and the biosynthesis of secondary metabolites. In vivo experiments have also concluded the inhibition of 6-phosphogluconate dehydrogenase to significantly reduce fitness in bloodstream forms in other organisms.
*Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Dimer
*Complex of proteins?: Dimer
*Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8 (TDR targets link)
*EC#: 1.1.1.44
*Link to BRENDA EC# page:
http://www.brenda-enzymes.info/enzyme.php?ecno=1.1.1.44
--Show screenshot of BRENDA enzyme mechanism schematic
*Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric Assay
-- link to Sigma/company page for assay:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/6phosphoglucdehydro74.pdf
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/mak038bul.pdf
-- links to assay reagents (substrates) pages:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/6phosphoglucdehydro74.pdf
--- List cost and quantity of substrate reagents, supplier, and catalog #
http://www.sigmaaldrich.com/catalog/product/sigma/mak038?lang=en®ion=US
Structure (PDB or Homology model)
-- PDB for Homology model: 1PGJ
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB:
---- Query Coverage: 96% coverage
---- Max % Identities: 73% identity
---- % Positives: 83% positive identity
---- Chain used for homology: A
Homology Model IMC:
Enter the Template PDB ID: 1PGJ
Identities 73% and Positives 57.9%
Enter the Molprobity scores for both the Template and the Model:
Template: Clash score = 14.28, Molprobity score = 2.77
Model: Clash score= 11.64, Molprobity score = 2.69
Current Inhibitors:
Acetyl-CoA, High concentrations of palmitoyl-CoA, ATP CoA , 1-Fluoro-2,4-dinitrobenzene (irreversible), Pyridoxal 5’-phosphate with respect to ZF (competitively) and NAD or NADP (noncompetitive), L. mesenteroides G6PD, unlike mammalian G6PDs, is unaffected by steroids.
Expression Information (has it been expressed in bacterial cells): Has been expressed in E. Coli cells
http://www.ncbi.nlm.nih.gov/pubmed/14698432
Purification Method: Nickel Column Chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
1 MSNDLGIIGLGVMGANLALNIAEKGFKVAVFNRTYAKTTSFLKEHESEKFAANLNGYETM
61 KEFAASLKKPRRAFILVQAGAATDSTIEQLKEVFENGDIIIDTGNANFKDQDKRAAQLES
121 QGLRFLGMGISGGEEGARKGPAFFPGGTPSVWEEVRPIVEAAAAKAEDGRPCVTFNGKGG
181 AGSCVKMYHNAGEYAVLQIWGEAYSALLAFGFDNDQIADVFESWKADGFLKSYMLDISIA
241 ACRAREATGNYLSEKVKDRIGSKGTGLWSAQEALEIGVPAPSLNMAVISRQMTMYKGERI
301 ANCKAFPNFPRGPSEEATDKSPNSPEAKKLYHAVSLCIIASYAQMFQCLRELDKVYGFGL
361 NLPATIATFRAGCILQGYLLGPMTKAFEENPNLPNLMDAFTKEIAAGLDDCRQILAKLTV
421 NTAVSLPVMMASLSYINAMYTETLPYGQLVSLQRDVFGRHGYERTDKDGRESFEWPALQX
*Length of your protein in Amino Acids: 480aa
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website: 52314.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence:
*GC% Content for gene (codon optimized): 60.4 %
*CDS Gene Sequence (codon optimized):
1 ATGTCTAACGACCTGGGTATCATTGGCCTGGGTGTTATGGGCGCGAACCTGGCGCTGAAC
61 ATCGCGGAAAAGGGCTTCAAGGTTGCGGTTTTTAACCGTACCTATGCGAAAACCACCAGC
121 TTCCTCAAAGAACACGAATCTGAAAAATTCGCTGCCAACCTGAACGGTTACGAAACCATG
181 AAGGAATTCGCGGCCTCCCTCAAAAAGCCGCGTCGTGCGTTCATCCTGGTCCAGGCGGGT
241 GCCGCGACGGACAGCACTATCGAACAGCTGAAAGAAGTATTCGAGAATGGTGACATCATC
301 ATCGACACTGGTAACGCGAACTTTAAGGACCAAGACAAACGTGCGGCACAGCTGGAATCT
361 CAGGGTCTGCGTTTCCTCGGTATGGGCATCTCTGGTGGCGAAGAAGGTGCGCGTAAAGGT
421 CCGGCGTTCTTTCCTGGCGGTACCCCGTCTGTCTGGGAAGAAGTCCGCCCGATCGTTGAA
481 GCAGCCGCAGCAAAAGCGGAAGACGGTCGTCCTTGCGTAACCTTCAATGGCAAAGGTGGT
541 GCGGGCTCTTGCGTTAAAATGTATCACAACGCGGGTGAATACGCGGTTCTGCAAATCTGG
601 GGTGAAGCGTACTCTGCGCTGCTGGCGTTCGGTTTCGACAACGACCAGATTGCGGACGTT
661 TTCGAGTCCTGGAAAGCCGACGGTTTCCTGAAGTCCTACATGCTGGACATCTCTATTGCG
721 GCGTGCCGTGCGCGCGAAGCAACCGGCAATTACCTGTCTGAGAAAGTGAAAGACCGTATC
781 GGTTCTAAAGGTACCGGTCTGTGGTCTGCACAGGAAGCGCTGGAAATCGGTGTTCCGGCA
841 CCGTCTCTGAATATGGCGGTTATCTCTCGTCAAATGACCATGTACAAGGGTGAGCGTATC
901 GCCAACTGCAAAGCGTTCCCGAACTTCCCGCGTGGTCCGTCTGAAGAAGCGACCGATAAA
961 TCTCCGAATTCTCCGGAAGCGAAAAAACTGTACCACGCCGTATCTCTGTGCATCATTGCG
1021 TCCTACGCCCAGATGTTCCAGTGCCTGCGTGAACTGGACAAAGTTTACGGCTTCGGTCTG
1081 AACCTGCCGGCGACCATCGCGACGTTCCGTGCGGGTTGTATCCTCCAGGGCTACCTGCTG
1141 GGTCCGATGACCAAAGCCTTTGAAGAAAATCCGAACCTCCCTAACCTGATGGACGCGTTC
1201 ACCAAAGAAATCGCGGCTGGTCTGGACGACTGTCGTCAGATCCTCGCGAAACTCACCGTT
1261 AACACGGCGGTTTCTCTCCCGGTCATGATGGCCTCTCTGAGCTACATCAACGCTATGTAT
1321 ACTGAAACCCTCCCATACGGCCAGCTCGTCTCTCTGCAGCGTGACGTATTTGGTCGTCAC
1381 GGTTATGAACGTACCGACAAAGATGGCCGTGAATCTTTCGAATGGCCGGCTCTGCAATAA
*GC% Content for gene (codon optimized): 54.2 %
Primer design results for pNIC-Bsa4 cloning:
1 ATGTCTAACGACCTGGGTATC 21
2 GTTCAGCGCCAGGTTCGCGCCCATAACACCCAGGCCAATGATACCCAGGTCGTTAGACAT 60
3 CGAACCTGGCGCTGAACATCGCGGAAAAGGGCTTCAAGGTTGCGGTTTTTAACCGTACCT 60
4 CAGATTCGTGTTCTTTGAGGAAGCTGGTGGTTTTCGCATAGGTACGGTTAAAAACCGCAA 60
5 CTTCCTCAAAGAACACGAATCTGAAAAATTCGCTGCCAACCTGAACGGTTACGAAACCAT 60
6 GCACGACGCGGCTTTTTGAGGGAGGCCGCGAATTCCTTCATGGTTTCGTAACCGTTCAGG 60
7 AAAAAGCCGCGTCGTGCGTTCATCCTGGTCCAGGCGGGTGCCGCGACGGACAGCACTATC 60
8 GATGATGATGTCACCATTCTCGAATACTTCTTTCAGCTGTTCGATAGTGCTGTCCGTCGC 60
9 CGAGAATGGTGACATCATCATCGACACTGGTAACGCGAACTTTAAGGACCAAGACAAACG 60
10 GAGGAAACGCAGACCCTGAGATTCCAGCTGTGCCGCACGTTTGTCTTGGTCCTTAAAGTT 60
11 CAGGGTCTGCGTTTCCTCGGTATGGGCATCTCTGGTGGCGAAGAAGGTGCGCGTAAAGGT 60
12 TTCTTCCCAGACAGACGGGGTACCGCCAGGAAAGAACGCCGGACCTTTACGCGCACCTTC 60
13 CCCGTCTGTCTGGGAAGAAGTCCGCCCGATCGTTGAAGCAGCCGCAGCAAAAGCGGAAGA 60
14 CCCGCACCACCTTTGCCATTGAAGGTTACGCAAGGACGACCGTCTTCCGCTTTTGCTGCG 60
15 GCAAAGGTGGTGCGGGCTCTTGCGTTAAAATGTATCACAACGCGGGTGAATACGCGGTTC 60
16 CGAACGCCAGCAGCGCAGAGTACGCTTCACCCCAGATTTGCAGAACCGCGTATTCACCCG 60
17 CGCTGCTGGCGTTCGGTTTCGACAACGACCAGATTGCGGACGTTTTCGAGTCCTGGAAAG 60
18 TAGAGATGTCCAGCATGTAGGACTTCAGGAAACCGTCGGCTTTCCAGGACTCGAAAACGT 60
19 TCCTACATGCTGGACATCTCTATTGCGGCGTGCCGTGCGCGCGAAGCAACCGGCAATTAC 60
20 GTACCTTTAGAACCGATACGGTCTTTCACTTTCTCAGACAGGTAATTGCCGGTTGCTTCG 60
21 ACCGTATCGGTTCTAAAGGTACCGGTCTGTGGTCTGCACAGGAAGCGCTGGAAATCGGTG 60
22 TTTGACGAGAGATAACCGCCATATTCAGAGACGGTGCCGGAACACCGATTTCCAGCGCTT 60
23 GGCGGTTATCTCTCGTCAAATGACCATGTACAAGGGTGAGCGTATCGCCAACTGCAAAGC 60
24 CGGTCGCTTCTTCAGACGGACCACGCGGGAAGTTCGGGAACGCTTTGCAGTTGGCGATAC 60
25 CGTCTGAAGAAGCGACCGATAAATCTCCGAATTCTCCGGAAGCGAAAAAACTGTACCACG 60
26 ACATCTGGGCGTAGGACGCAATGATGCACAGAGATACGGCGTGGTACAGTTTTTTCGCTT 60
27 CGTCCTACGCCCAGATGTTCCAGTGCCTGCGTGAACTGGACAAAGTTTACGGCTTCGGTC 60
28 CAACCCGCACGGAACGTCGCGATGGTCGCCGGCAGGTTCAGACCGAAGCCGTAAACTTTG 60
29 CGTTCCGTGCGGGTTGTATCCTCCAGGGCTACCTGCTGGGTCCGATGACCAAAGCCTTTG 60
30 GTGAACGCGTCCATCAGGTTAGGGAGGTTCGGATTTTCTTCAAAGGCTTTGGTCATCGGA 60
31 CCTGATGGACGCGTTCACCAAAGAAATCGCGGCTGGTCTGGACGACTGTCGTCAGATCCT 60
32 TGACCGGGAGAGAAACCGCCGTGTTAACGGTGAGTTTCGCGAGGATCTGACGACAGTCGT 60
33 CGGTTTCTCTCCCGGTCATGATGGCCTCTCTGAGCTACATCAACGCTATGTATACTGAAA 60
34 ACGCTGCAGAGAGACGAGCTGGCCGTATGGGAGGGTTTCAGTATACATAGCGTTGATGTA 60
35 TCGTCTCTCTGCAGCGTGACGTATTTGGTCGTCACGGTTATGAACGTACCGACAAAGATG 60
36 TTATTGCAGAGCCGGCCATTCGAAAGATTCACGGCCATCTTTGTCGGTACGTTCATAA 58
Primer design results for 'tail' primers (this is just 2 sequences):
Forward:
5' TACTTCCAATCCATGTCTAACGACCTGGGTATCATTGGC 3'
Reverse:
5' TATCCACCTTTACTGTTATTGCAGAGCCGGCCAT 3'